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. 2007 May 1;403(3):483-92.
doi: 10.1042/BJ20061182.

Distinct roles of the C2A and the C2B domain of the vesicular Ca2+ sensor synaptotagmin 9 in endocrine beta-cells

Florence Grise  1 , Nada Taib, Carole Monterrat, Valérie Lagrée, Jochen Lang
Affiliations

Distinct roles of the C2A and the C2B domain of the vesicular Ca2+ sensor synaptotagmin 9 in endocrine beta-cells

Florence Grise et al. Biochem J. .

Abstract

Synaptotagmins form a family of calcium-sensor proteins implicated in exocytosis, and these vesicular transmembrane proteins are endowed with two cytosolic calcium-binding C2 domains, C2A and C2B. Whereas the isoforms syt1 and syt2 have been studied in detail, less is known about syt9, the calcium sensor involved in endocrine secretion such as insulin release from large dense core vesicles in pancreatic beta-cells. Using cell-based assays to closely mimic physiological conditions, we observed SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-independent translocation of syt9C2AB to the plasma membrane at calcium levels corresponding to endocrine exocytosis, followed by internalization to endosomes. The use of point mutants and truncations revealed that initial translocation required only the C2A domain, whereas the C2B domain ensured partial pre-binding of syt9C2AB to the membrane and post-stimulatory localization to endosomes. In contrast with the known properties of neuronal and neuroendocrine syt1 or syt2, the C2B domain of syt9 did not undergo calcium-dependent membrane binding despite a high degree of structural homology as observed through molecular modelling. The present study demonstrates distinct intracellular properties of syt9 with different roles for each C2 domain in endocrine cells.

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Figures

Figure 1
Figure 1. Expression of syt2 and syt9 constructs
(A) Schematic diagram representing the different syt9 and syt2 constructs used: syt2C2AB (101–422), syt9C2AB (77–386), syt9C2A (77–233) and syt9C2B (215–386). Mutation of the aspartic acid residue to an asparagine residue in the C2A and/or the C2B domains gave rise to D145N, D197N, D199N, D330N and D332N. All constructs are C-terminally tagged with a fluorescent protein (FP). (B) Expression of the constructs in HIT-T15 cells. Cells were transfected with the different variants. After transfection (72 h), cells were harvested and 20 μg of total proteins were separated by SDS/PAGE and immunoblotted with anti-syt2, anti-syt9 or anti-eGFP antibodies. The lane number corresponds to the construct number.
Figure 2
Figure 2. Membrane binding of syt9C2AB–eGFP in HIT-T15 cells
(A) After transfection (72 h) with syt9C2AB–eGFP, HIT-T15 cells were incubated for 5 min at 37 °C in the presence of Ca2+ (2 mM CaCl2 supplemented with 10 μM ionomycin) or in absence of Ca2+ (2 mM EGTA) and fractioned by ultracentrifugation at 55000 rev./min. Distribution of syt9C2AB–eGFP in supernatants (c) and membrane pellets (m) were analysed by Western blot using antibodies against syt9 and against the transmembrane protein syntaxin 1 as a control for fractionation. The distribution was quantified by densitometry as given by the histogram (on the right-hand side), Means±S.D.s are indicated (n=8). (B) Transfected cells were incubated in the absence or in the presence of Ca2+ supplemented with 0.5 M KCl. Fractionation and analysis were performed as described above (n=3). (C) HIT-T15 cells were co-transfected with syt9C2AB–eGFP and plasmids coding for botulinum neurotoxins BoNT/C and BoNT/E. Translocation and the effect of each toxin were analysed by immunoblotting using anti-syt9, anti-syntaxin1 or anti-SNAP-25 antibodies. Errors bars represent the S.D. (n=3); *2P<0.05 as compared with membranes.
Figure 3
Figure 3. The C2A but not the C2B domain is responsible for Ca2+-dependent binding of syt9 to membranes
(A) HIT-T15 cells expressing PKC-C2α–eGFP, syt2C2AB–eGFP and syt9C2AB–eGFP were incubated for 5 min at 37 °C in the presence (2 mM CaCl2 and 10 μM ionomycin) or the absence (2 mM EGTA) of Ca2+. The subsequent fractionation was analysed by Western blot using anti-GFP, anti-syt2 or anti-syt9 antibodies for PKC-C2α–eGFP, syt2C2AB–eGFP and syt9C2AB–eGFP respectively. Histograms on the right-hand side represent the distribution of the protein quantified by densitometry. Errors bars represent the S.D (n=3); *2P<0.05 as compared with membranes. (B) The same experiments in (A) were performed on HIT-T15 cells transfected with syt9C2A–eGFP and syt9C2B–eGFP. Syt9C2A–eGFP was revealed with the anti-syt9 antibody that detected the fluorescent protein (indicated by the arrowhead) and the endogenous protein. Syt9C2B–eGFP was revealed with the anti-GFP antibody as it does not contain the epitope for the anti-syt9 antibody. (C) Syt9C2AB–eGFP and its mutants were detected using anti-syt9.
Figure 4
Figure 4. Molecular simulation of the syt9C2B domain
Ribbon diagrams are shown. (A) Syt1C2B according to the crystal structure 1UOW. (B) C2B domain of syt9 after 3 ns simulation. The loops 1 and 3 (L1 and L3), the mutation D330N/D332N (*), the unstructured part corresponding to the β-sheet 4 in syt1 and the shortened α-helix H2 are indicated. Views are given to highlight the differences between the structures.
Figure 5
Figure 5. Stimulation of living cells by low micromolar Ca2+ translocates the C2A, but not the C2B domain, of syt9
(A) MIN6 cells expressing syt9C2AB–eGFP, syt9C2A–eGFP or syt9C2B–eGFP were grown on coverslips and stimulated by pressure ejection of buffer containing 10 μM free Ca2+ supplemented with 30 μM digitonin. Cells were imaged at 37 °C by time-lapse microscopy. Images a, c and e were taken at 0 s and, b, d and f 3 s after stimulation. (B) Membrane binding affinity of syt9 variants. Experiments were performed in MIN6 cells as described in (A). Defined concentrations of free Ca2+ supplemented with digitonin were used to stimulate the cells.
Figure 6
Figure 6. Syt9C2AB–eGFP but not syt9C2ABD330N/D332N–eGFP distributes to intracellular structures after stimulation with Ca2+
MIN6 cells expressing syt9C2AB–eGFP (A, B and C) or syt9C2ABD330N/D332N–eGFP (E, F and G) were stimulated by 5 mM CaCl2 and 10 μM ionomycin. The distribution of the two fusion proteins was imaged and the three panels represent the distribution of the proteins at 0 s (A and E), 10 s (B and F) and 20 s after stimulation (C and G). GFP fluorescence was quantified in two different areas corresponding to plasma membrane (zone 1) and to the intracellular space (zone 2) (D and H). Fo, fluorescence at t=0 s; Ft, fluorescence at t. Images are representative of at least ten independent experiments.
Figure 7
Figure 7. Syt9C2AB redistributes to endosomes
MIN6 cells transfected with indicated constructs were incubated for 10 min at room temperature in the presence of Ca2+ (5 mM CaCl2 and 10 μM ionomycin) to allow the translocation of syt9C2AB-FP [eGFP or S (Strawberry)] to the plasma membrane and to intracellular structures. Images corresponding to the different channels are given in the left-hand panels and middle panels; co-localization is indicated by ‘MERGE’ in the right-hand panels. EEA1, early endosomal antigen. (A) Cells were subsequently fixed and co-localization of the FP constructs analysed by confocal microscopy. (B) Living cells observed by videomicroscopy 10 min after stimulation. (C) Percentage of co-localization given as obtained from at least five experiments.
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